Ligation of single stranded (ss) DNA oligonucleotides splinted by complementary RNA is an essential step in techniques such as RNA-mediated annealing, selection, and ligation (RASL). T4 DNA ligase has been used for RASL as well as for other RNA analysis and detection techniques such as molecular inversion probes, modified ligase chain reactions and ligase detection reactions (for example, Yeakley, et al., Nat Biotechnol., 20(4):353-8 (2002), Bullard and Bowater, Biochem. J., 398(1):135-44 (2006); Li, et al., Curr Protoc Mol Biol. April; Chapter 4: Unit 4.13.1-9 (2012); US published application No. 2011/0092375, U.S. Pat. No. 7,361,488; Nilsson, et al., Nature Biotechnology, 18:71 (2000); Nilsson, et al., Science, 265, 2085-2088 (1994); Barany, PCR Methods Appl., 1:5-16 (1991); Landegren, Bioessays, 15:761-765 (1993); Wiedmann, et al., PCR Methods Appl., 3:S51-64 (1994); Nilsson, et al., Nat Genet., 16:252-255 (1997); Baner, et al., Nucleic Acids Res., 26:5073-5078 (1993); Hardenbol, et al., Nature Biotechnol., 21:673-678 (2003); and Landegren, Methods Cell Biol., 75:787-797 (2004)).
T4 DNA ligase works poorly requiring, for example, long incubation times, high concentrations of ligase, and low ATP concentrations to overcome the preferential formation of adenylated DNA side product to accomplish ligation.
T4 RNA ligase was tested as an alternative choice for joining DNA strands hybridized to an RNA template or splint (U.S. Pat. No. 6,368,801). The NAD+ dependent ligase from Melanoplus sanguinipes entomopoxvirus was reported to have a ligation activity for DNA hybridized to RNA similar to T4 DNA ligase but only in the presence of Mn2+ (Lu, et al., Biocimica et Biophysica Acta, 1701:37-48 (2004)). Sriskanda, et al., Nucleic Acid Research, 26 (15):3536-3541 (1998) reported PBCV-1DNA ligase from Chlorella where experimental data showed that this ligase could ligate oligonucleotides on a DNA template or DNA splint but could not ligate oligonucleotides on an RNA template or RNA splint. These results were explained by crystal structure studies where the authors showed that PBCV-1 ligase forced the substrate into an RNA-type A-form helix on one side of a nicked substrate, but required a DNA-type B-form helix on the side of the nick providing the 5′ phosphate (Ho, et al., J. Viral., 71(3):1931 (1997); Sriskanda, et al., (1998); Nair, et al., Nat. Struct. Mol. Biol., 14:770-778 (2007)). Similar results were reported in crystal structures of the NAD-dependent E. coli DNA ligase (Nandakumar, Mol. Cell, 26:257-271 (2007)) and human DNA ligase 1 (Pascal, et al., Nature, 432:473-478 (2004)) leading to a conclusion that these ligases could not accept RNA-splinted DNA as ligation substrates.